Artificial tissue constructs comprising alveolar cells and methods for using the same

ABSTRACT

The present invention comprises artificial tissue constructs that serve as in vitro models of mammalian lung tissue. The artificial tissue constructs of the present invention comprise functionally equivalent in vitro tissue scaffolds that enable immunophysiological function of the lung. The constructs can serve as novel platforms for the study of lung diseases (e.g., interstitial lung diseases, fibrosis, influenza, RSV) as well as smoke- and smoking-related diseases. The artificial tissue constructs of the present invention comprise the two components of alveolar tissue, epithelial and endothelial cell layers.

CROSS REFERENCE TO RELATED CASES

This application is a divisional of U.S. application Ser. No.12/173,921, filed Jul. 16, 2008, which application claims the benefit ofU.S. Provisional Application Ser. No. 60/949,944, filed Jul. 16, 2007,which are incorporated by reference herein in their entirety.

FIELD OF THE INVENTION

The present invention comprises artificial or man-made models ofmammalian lung tissue and methods of using the same. The artificialtissue constructs of the present invention are functionally equivalentto in vitro lung tissue scaffolds that enable reliable and predictablemodeling of human immunophysiological responses to particularconditions, environments and/or factors. The artificial tissueconstructs can serve as novel platforms for the study of lung diseases(e.g., interstitial lung diseases, fibrosis, influenza, RSV,tuberculosis, anthrax, allergies), smoke- and smoking-related diseases,and the effects of particulate inhalation. The artificial tissueconstructs of the present invention provide a structural basis for twocomponents of alveolar tissue, namely an epithelial cell layer and anendothelial cell layer. These layers include appropriate cells,cytokines, growth factors, and extracellular matrix (ECM) materials.Methods are also provided that enable production of biocompatible,cellular, heterogeneous engineered tissue constructs (ETCs) that mimicthe bronchoalveolar regions and alveolar sacs of mammalian lung tissue.

BACKGROUND OF THE INVENTION

One fifth of global mortality has been reported to be due to infectiousdisease. Within this, respiratory diseases alone account for about fourmillion deaths per year (Girard et al. (2005) Vaccine 23, 5708-24).However, because of the expense of development, coupled with uncertaintyregarding efficacy, only about 2% of the global pharmaceutical budget isallocated to development of new prophylactic vaccines (Kiney & Girard(2005) Vaccine 23, 5705-7). Given the scope of the worldwide healthproblems caused by known and emerging infectious diseases, andadditionally, the potential of novel biological warfare pathogens, it isimportant that novel strategies of rapid vaccine evaluation be developedand implemented.

Lung Structure and Function

The mammalian lung is an organ within the thorax where passive diffusionof oxygen and carbon dioxide occur across a thin, two-cell-thick tissue.Normal lung function is to exchange oxygen from the air with carbondioxide in the blood. Only two cell layers are interposed between theair and the blood, with a small amount of ECM serving as a scaffold.

The respiratory mucosa is the largest organ system directly open to theoutside environment, with a surface area of 60-80 m². It serves as apoint of entry for nutrient gases, along with microorganisms andparticulate antigens that can trigger a multitude of respiratory immuneresponses. The first and the last point of contact occur at thenasopharynx and the alveolar regions of the lung, respectively.

The alveolus is the site where gaseous exchange occurs between thealveolar type I cells (respiratory epithelial cells), alveolar type IIcells, and the vascular endothelial cells of the surroundingcapillaries. These two cell layers prevent the direct mixing of air andblood, yet provide throughout the lung alveoli a massive surface areafor gaseous exchange to occur. In an adult human, this surface area isabout 60 m² during full expiration and about 80 m² during fullinspiration.

Many pathophysiological conditions result in the direct increase orreduction of pulmonary mass, leading to a decrease in gaseous exchange.Non-malignant and malignant primary and metastatic lung tumors areprincipal reasons for permanent decreases in pulmonary function. Othercauses of decreased pulmonary function include viral and bacterialpneumonias, trauma, fibrosis, and idiopathic disease.

The alveoli are tiny air sacs, the walls of which are covered withcapillaries across which oxygen and carbon dioxide readily diffuse andare transferred into and out from the blood, respectively. This exchangeis essential to survival and is the key function of the lungs.

The alveolus is a site where only two cell layers are interposed betweenthe air and the blood, with a small amount of ECM serving as amembranous scaffold. Gaseous exchange occurs across the alveolar wall,comprising alveolar type I and type II cells (squamous respiratoryepithelial cells) and the vascular endothelial cells of the surroundingcapillaries.

Alveoli have fragile, thin walls, which are easily damaged. Breakage ofthese walls makes the oxygen-carbon dioxide diffusion much lessefficient. The bronchial tree distributes the air throughout the lung tothe individual alveoli. Once damaged, the bronchioles tend to collapse,trapping stale air in the isolated sacs and not letting fresh air in,leading to atelectasis.

Emphysema permanently enlarges and irreversibly damages the alveoli. Itdamages the ends and walls of the smallest bronchioles (the tinybreathing tubes that branch off from the trachea and bronchi), anddiminishes pulmonary elasticity.

As alveoli and bronchial tubes are destroyed in pathophysiologicalconditions, progressively more air is required to provide the sameamount of oxygen to the blood via the parts of the lung that are stillfunctioning. This need for more air eventually leads to lungover-inflation. As the lung over-expands, it gradually enlarges,completely filling the maximum thoracic cage volume and causing a senseof shortness of breath. Because the lung can no longer expand orcontract as completely as before, stale air left in the lung is nevercompletely replaced with fresh air, resulting in poor gas exchange. Thecombination of a larger, less elastic lung and damaged, non-functioningtissue means that the air flow out of the lung is much slower, resultingin the feeling of an obstructed airway.

Many lung diseases that cause a narrowing of the respiratory airways(e.g., chronic bronchitis, asthma) can contribute to the onset ofemphysema, but smoking is a common cause. In addition to theirreversible damage smoking causes to lung tissue, it causesinflammation of the lungs, which resolves only when smoking stops.Smoking also stresses the natural antioxidant defense system of thelung, allowing free radicals to damage lung tissue at the cellularlevel.

Additionally, irritants contained in tobacco smoke tend to inhibitactivity of the cilia of the airways. These cilia ordinarily function toexpel foreign matter and mucus from the lung. Without their activity, itbecomes difficult or impossible to cough up the mucus that accompaniespneumonia and other lung infections. Cigarette smoke can temporarilyparalyze the cilia. Smoking-induced emphysema usually becomes apparentafter age 50.

The deposition of an inhaled particle in the lung has been linked to itssize. For example, the upper respiratory mucosa is the first anatomicbarrier where particles from ˜5 to ˜10 μm are deposited, while ˜0.2 to˜2-μm sized particles are deposited in the lower mucosal alveolarregion.

At the molecular level, the initial responses to such foreign particlesinclude opsonizing agents (e.g., collectins), activation of cytochromeP450s, complement, lysozymes, anti-bacterial peptides (e.g., defensins),mannose-binding proteins, and interferons. Phagocytic cells activated atprimary antigen deposition sites include alveolar macrophages andnatural killer (NK) cells, which have the ability to recognize andneutralize infected cells, through recognition of bacterial and viralfeatures. The adaptive responses trigger T and B lymphocytes that buildimmunological memory to subsequent challenges. This process is primarilyregulated by antigen-presenting cells (APCs), such as macrophages,dendritic cells (DCs), and Type II epithelial cells. The T cell receptor(TCR) on the surface of the T lymphocyte is only activated by sensingmajor histocompatibility complex (MHC) molecules containing processedantigenic peptides on the surface of APCs. T cell responses aredependent on cytokines produced and functional effects afterencountering antigen-specific T cells.

Respiratory epithelial cells have been shown to be responsive tolipopolysaccharides (Koyama et al. (1991) J. Immunol. 147, 4293-301;Diamond & Bevins (1994) Chest 105(3 Suppl), 51S-52S), muramyl dipeptides(Lopez-Boado et al. (2000) J. Cell Biol. 148, 1305-15; Diamond et al.(2000) Infect Immun. 2000 January; 68(1):113-9; Bevins (2003) Contrib.Microbiol. 10, 106-48), and lipoteichoic syncytial acid (Wagner et al.(1999) Am. J. Respir. Cell Mol. Biol. 20, 769-76; Diamond et al. (2000)Infect Immun. 2000 January; 68(1):113-9). They express Toll-likereceptors (Holgate (2007) Trends Immunol. 28, 248-51; Bals & Hiemstra(2004) Eur. Respir. J. 23, 327-33; Becker et al. (2000) J. Biol. Chem.275, 29731-6), TNF receptors (Levine (1995) J. Investig. Med. 43, 241-9;Nettesheim & Bader (1996) Toxicol. Lett. 88, 35-7), and demonstrateup-regulation of host defense genes, such as like MUC2, MUC5C, hBD2, andLL37/CAP18 (Becker et al. (2000) J. Biol. Chem. 275, 29731-6; Agerberthet al. (1999) Am. J. Respir. Crit. Care. Med. 160, 283-90; Bartman etal. (1998) J. Pathol. 186, 398-405; Yoon & Park (1998) Rhinology 36,146-52; Dohrman et al. (1998) Biochim. Biophys. Acta 1406, 251-9; Li etal. (1997a) J. Pathol. 181, 305-10; Li et al. (1997b) Proc. Natl. Acad.Sci. USA 94, 967-72). Respiratory epithelium also produces interleukins(IL-1, IL-5, IL-6, IL-8), RANTES (regulated upon activation, normal Tcell-expressed, and secreted), endothelin, granulocyte-monocyte colonystimulating factor (GM-CSF), transforming growth factor beta (TGF-β),interferon-γ-induced protein (IP-10), interferon-inducible T-cellα-chemoattractant (I-TAC), and γ-interferon-inducible T cellchemoattractant (Chung (2006) Curr. Drug Targets 7, 675-81; Prescott(2003) J. Paediatr. Child Health 39, 575-9; Holt & Stumbles (2000) J.Allergy Clin. Immunol. 105, 421-429).

As part of the adaptive immune response, memory T cells are constantlycirculating through the lung parenchyma including alveolar spaces viawell characterized lymphocyte homing mechanisms (Wardlaw et al. (2008)Clin. Exp. Allergy 35, 4-7). As the lung is considered a tertiarylymphoid organ (Grigg & Riedler (2000) Am. J. Respir. Crit. Care 162,52-5), it contains large numbers of memory T cells in all compartmentsof the respiratory tract, with the largest number in the lung, migratingin through the post capillary venules under low hydrodynamic pressures.These memory T cells migrate specifically to the organ of their cognateantigen initiation. Much fewer naïve T cells enter the lung as ageincreases and only respond to newly seen antigens. These naïve T cellsmust enter the lung alveolar parenchyma via high endothelial venulesunder higher vascular pressures and faster flow. Unregulated T cellemigration into alveolar spaces and respiratory parenchyma may be a keyfactor for the formation of asthma (Bedoff et al. (2008) Annu. Rev.Immunol. 26, 205-32).

Respiratory Delivery Route

Advances in respiratory mucosal delivery have been driven by thenon-invasive, highly absorptive properties of the respiratory route.Compared with the oral route, respiratory delivery offers a lack ofdigestive enzymes or mechanical forces, along with thin walls and ahighly absorptive vascularized surface area for improved systemicdelivery.

For vaccine or drug delivery, nebulizers and powder inhalers allowdeposition of therapeutics in specific sites of the lung (Byron (2004)Proc. Am. Thorac. Soc. 1, 321-8; Laube (2005) Respir. Care 50, 1161-76).The potential for aerosolized vaccine delivery for influenza andmeasles, in addition to delivery of peptides and small molecule drugshas been explored with some success in asthma, chronic obstructivepulmonary disease (COPD), migraine, and diabetes-related therapeutics(Laube (2005) Respir. Care 50, 1161-76; Kennedy (1991) Drugs 42, 213-27;Ilium (2002) Drug Discov. Today 7, 1184-9; Sullivan et al. (2006) ExpertOpin. Drug Deliv. 3, 87-95). A prominent FDA-approved aerosolizedvaccine is FluMist®, for seasonal flu. However, our limitedunderstanding of the mechanistic details of the respiratory drugdelivery has hampered the development of aerosolized therapeutics.

Other Respiratory Immunology/Toxicology Models: In Vivo and In VitroApproaches

Since the 1970s there has been interest in developing in vivorespiratory models to study human immunology (e.g., Chowhan & Amaro(1976) J. Pharm. Sci. 65, 1669-72; Torkelson et al. (1976) Am. Ind. Hyg.Assoc. J. 37, 697-705; Belshe et al. (1977) J. Med. Virol. 1, 157-62;Saffiotti (1978) Environ. Health Perspect. 22, 107-13; Schanker (1978)Biochem. Pharmacol. 27, 381-5). Previous in vitro lung models includedthose based on the Transwell™ permeable support device or similarconstruct, comprising an endothelial cell layer, an epithelial celllayer, and an artificial microporous membrane, having pores therein,disposed between and in direct contact with the endothelial cell layerand the epithelial cell layer such that the membrane has an endothelialside and an epithelial side (see, e.g., U.S. Pat. No. 5,750,329; Weppleret al. (2006) Exp. Lung Res. 32, 455-82; Birkness et al. (1995) Infect.Immun. 63, 402-9; Birkness et al. (1999) Infect. Immun. 67, 653-658).

U.S. Pat. No. 5,750,329 describes a method for constructing anartificial lung system, comprising placing an artificial microporousmembrane, having pores therein, into a vessel having a bottom andsupporting the membrane a distance from the bottom of the vessel tocreate an upper and lower chamber in the vessel such that the membranehas an endothelial side facing into the lower chamber of the vessel andan opposite epithelial side facing into the upper chamber of the vessel;placing endothelial cells into the upper chamber of the vessel underconditions such that the endothelial cells form a confluent layer ofcells on the epithelial side of the membrane; and placing alveolarepithelial cells into the upper chamber of the vessel under conditionssuch that the endothelial cells migrate through the pores in themembrane and attach to the endothelial side of the membrane to form aconfluent layer of the endothelial cells on the endothelial side of themembrane in the lower chamber and the alveolar epithelial cells form aconfluent layer of the epithelial cells on the epithelial side of themembrane in the upper chamber.

While in vivo animal models have been developed, variations in animalsize, species, and differences in therapeutic distribution have resultedin inconsistencies in reported findings by various research groups usingsuch models. Most in vivo models require destructive means for animaldosing using test compounds and vaccines, and blood and tissue isolationthat also depend on animal handling/surgical protocols that are underscrutiny (Schanker & Hemberger (1984) Pharmacology 28, 47-50; Schanker &Hemberger (1983) Biochem. Pharmacol. 32, 2599-601; Schanker et al.(1986a) Pharmacology 32, 176-80; Schanker et al. (1986b) Drug Metab.Dispos. 14, 79-88; Schanker (1978) Biochem. Pharmacol. 27, 381-5;Hemberger & Schanker (1983a) Drug Metab. Dispos. 11, 73-4; Hemberger &Schanker (1983b) Drug Metab. Dispos. 11, 615-6; Brown & Schanker (1983a)Drug Metab. Dispos. 11, 355-60; Brown & Schanker LS (1983b) Drug Metab.Dispos. 11, 392-3; Lin & Schanker (1983a) Drug Metab. Dispos. 11, 75-6;Lin & Schanker (1983b) Drug Metab. Dispos. 11, 273-4; Mobley & Hochhaus(2001) Drug Discov. Today 6, 367-375, Widdicombe (1997) J. Appl.Physiol. 82, 3-12; Flecknell (2002) ALTEX 19, 73-8; Abbott (2005 Nature438, 144-6). Generally, the cost and time required for in vivo animalmodels along with physiological variations in the selected animal andhuman species (Li et al. (2007) Exp. Lung Res. 33, 227-44; Cao et al.(2007) Toxicol. Lett. 171, 126-37; Denham et al. (2007) Am. J. Physiol.Lung Cell. Mol. Physiol. 292, L1241-7; Carter et al. (2006) J. Occup.Environ. Med. 48, 1265-78) makes them less attractive than in vitromodels for many purposes.

Compared with in vivo approaches, in vitro models are considered morehumane, cost effective, are generally simpler to execute, and can usemore physiologically relevant human cells. Various approaches have beenadopted to develop in vitro respiratory system models using epithelialcell lines. However, the intrinsic differences between the functionalityof these cell lines and native primary cells make these systems lessthan ideal. Several lung cell lines have been reported, includingcarcinoma-derived epithelial cell lines, such as A549 (alveolar),Calu-1, Calu-3, Calu-6, H441, HBE1, and A427. Normal tissue-derivedtransformed cell lines include 16HBE14o- (bronchial),9HTE16o-(tracheal), 1HAEo-(tracheobronchial), BEAS-2B (bronchial), andCF/T43 (nasal). However, undesirable changes in cell linecharacteristics over passage culture have been reported, such asmorphology, growth rates, protein expression, permeability, andsignaling (ATCC Technical Bulletin no. 7, Passage number effects on celllines. 2007 1-3; Esquenet et al. (1997) J. Steroid Biochem. Mol. Biol.62, 391-9; Briske-Anderson et al. (1997) Proc. Soc. Exp. Biol. Med. 214,248-57; Chang-Liu & Woloschak (1997) Cancer Lett. 113, 77-86; Yu et al.(1997) Pharm. Res. 14, 757-62, Sambuy et al. (2005) Cell Biol. Toxicol.21, 1-26; Wenger et al. (2004) Biosci. Rep. 24, 631-9).

Such intrinsic differences can also be of concern when developing invitro screening systems for therapeutics and pathogens. The need forhighly functional primary lung epithelial cells has led to developmentof a number of primary cell isolation methods for mouse (Corti et al.(1996) Am. J. Respir. Cell Mol. Biol. 14, 309-15), rat (Goodman &Crandall (1982) Am. J. Physiol. 243, C96-100; Cheek et al. (1989) Exp.Cell Res. 184, 375-87) rabbit (Shen et al. (1999) Pharm. Res. 16,1280-7), pig (Steimer et al. (2006) Pharm. Res. 23, 2078-93), and humanlung tissue (Bur et al. (2006) Eur. J. Pharm. Sci. 28, 196-203; Elbertet al. (1999) Pharm. Res. 16, 601-8). However, differences betweentissue responses between species have been noted, a possible drawback tousing primary animal lung cells (Denham et al. (2007) Am. J. Physiol.Lung Cell. Mol. Physiol. 292, L1241-7; Carter et al. (2006) J. Occup.Environ. Med. 48, 1265-78).

Viral pathogens that cause respiratory disease include common flu orinfluenza (A or B; Orthomyxoviridae family), respiratory syncytialvirus, human parainfluenza viruses (HPIVs; paramyxovirus family),metapneumovirus (hMPV; family Paramyxoviridae), adenoviruses,rhinoviruses, parainfluenza viruses, coronaviruses, coxsackievirus, andherpes simplex virus. Respiratory disease bacterial pathogens includeYersinia pestis, Bacillus anthracis, Escherichia coli, Francisellatularensis, Staphylococcus aureus Group A beta-hemolytic streptococci(GABHS), group C beta-hemolytic streptococci, Corynebacteriumdiphtheriae, Neisseria gonorrhoeae, Arcanobacterium haemolyticum,Chlamydia pneumoniae, Mycoplasma pneumoniae, Streptococcus pneumoniae,Haemophilus influenzae, Moraxella catarrhalis, Bordetella pertussis, andBordetella parapertussis.

There is a continuing need for a predictive, reproducible in vitro modelsystem based on lung immunophysiology and function that would enable thestudy of a broad spectrum of respiratory disease pathogenesis andassociated therapies. There is also a continuing need for in vitroimmunological approaches for accurately predicting human immunologicalresponses. The artificial tissue constructs of the present invention,with their use of three-dimensional (3D) tissue engineering and advancedcell biology, combined with modern bio-fabrication and bioreactortechniques, provide such a model system.

BRIEF DESCRIPTION OF THE INVENTION

The present invention comprises artificial tissue constructs comprisingtwo layers of cells, wherein one layer of cells is positioned atop theother layer of cells, and wherein one of the layers of cells comprisesalveolar primary epithelial cells and the other layer of cellscomprising alveolar primary endothelial cells. The present inventionalso comprises methods of determining whether a test compound hasimmunological activity, comprising culturing an artificial tissueconstruct of the present invention in the presence of a test compound,and determining the effect the test compound has on an immunologicalactivity of the artificial tissue construct Immunological activities ofthe artificial tissue construct include immunoglobulin generation,chemokine generation and cytokine generation. The present inventionfurther provides methods for preparing the artificial tissue constructsof the present invention.

The present invention also provides methods of preparing heterogeneoustissue constructs, comprising cells on the surfaces of a biocompatiblemembrane that mimic a vascular endothelium and a respiratory epithelium.In preferred embodiments the biocompatible membrane is selected from thegroup consisting of extracellular matrix, collagen, laminin,proteoglycan, vitronectin, fibronectin, poly-D-lysine andpolysaccharide.

In other embodiments, other biocompatible membrane can be used to createan alveolar model. Additionally, the ECM structure can comprisedifferent bio-material formulations, thicknesses, porosities, andcross-linking agents.

The artificial tissue constructs of the present invention can be used aspart of in vitro diagnostic immunological methods for testing theeffects of, for example, therapeutics, vaccines, particulates, andrespiratory disease pathogens in the upper and lower airways. Usingartificial tissue constructs of the present invention, comprisingprimary human cells, correlative responses can be determined fromclinical studies and the supporting literature.

By increasing the accuracy of the artificial tissue constructs used inpre-clinical vaccine testing, it is possible to accelerate the rate ofdrug testing, increase the probability that any particular drugcandidate will be successful in human trials, and augment the collectionof more predictive data that will aid in pre-clinical design andformulation of therapeutics.

An embodiment of the present invention comprises an engineered 3Dartificial tissue construct that mimics respiratory immunologicalfunction, and is superior to 2D cell representations and more relevantthan animal models in assessing human respiratory immune responses.

Cell-based in vitro respiratory mucosal models for immunologicalevaluation have been under-used in the past because of limitations inmaintaining primary cell function and the disparity of responses ofintrinsically different cell lines. Embodiments of the present inventioninclude the fabrication and functional testing of in vitro grownartificial tissue constructs, comprising primary human epithelial andendothelial cells from alveolar regions, that can be used to study theeffects of and faithfully mimic the responses to, for example, airbornepathogens and human respiratory immunology. Variations in the thicknessof the interposed extracellular matrix along with differing epithelialand submucosal cell types, will reproduce the different areas in thelung.

Historically, most in vitro models have used single or multiple layersof epithelial cells. The bilayer artificial tissue constructs of thepresent invention comprise an epithelium and an endothelium,representing the epithelial mucosa and the local tissuemicrovasculature. Autologous blood cells can be used to mimic thenatural environment.

Embodiment of the present invention addresses the disadvantages in usinganimal models, because of physiological differences. The artificialtissue constructs of the present invention comprise native human cellsof appropriate phenotypes from the alveolar regions.

The artificial tissue constructs of the present invention can be used topredict human physiological/immunological responses in an in vitrosetting. Further, they can be used to assess responses to viral (e.g.,influenza, respiratory syncytial virus) and bacterial (e.g., E. coli andS. aureus) exposure. The effects of, for example, immunosuppressivecorticosteroid hormones (e.g., dexamethasone, hydrocortisone,alclometasone, amcinonide, beclometasone, betamethasone, budesonide,ciclesonide, clobetasol, clobetasone, clocortolone, cloprednol,cortivazol, deflazacort, deoxycorticosterone, desonide, desoximetasone,diflorasone, diflucortolone, difluprednate, fluclorolone,fludrocortisone, fludroxycortide, flumetasone, flunisolide, fluocinoloneacetonide, fluocinonide, fluocortin, fluocortolone, fluorometholone,fluperolone, fluprednidene, fluticasone, formocortal, halcinonide,halometasone, hydrocortisone aceponate, hydrocortisone buteprate,hydrocortisone butyrate, loteprednol, medrysone, meprednisone,methylprednisolone, methylprednisolone aceponate, mometasone furoate,paramethasone, prednicarbate, prednisone, prednisolone, prednylidene,rimexolone, tixocortol, triamcinolone, ulobetasol) and environmentalirritants (e.g., diesel particles) and potentially carcinogenicpolycyclic hydrocarbons (e.g., 3-methylcholantherene, β-nitrophenol) canalso be evaluated.

Embodiments of the present invention comprise bilayer alveolar mucosalmodels, comprising primary alveolar epithelial and endothelial cells,optionally cultured with extracellular matrix proteins, that promote adesirable type-II epithelial cell phenotype and biological function. Tomake the artificial tissue constructs, both lung and other tissue andcell samples are preferably from the same person or animal.

In preferred embodiments of the present invention, the bilayerartificial tissue constructs comprise human primary alveolar epithelialand human endothelial cells cultured with extracellular matrix proteinsthat promote a desirable type-II epithelial cell phenotype andbiological function. To make the constructs, both lung and other tissueand cell samples are preferably from the same person.

The bilayer alveolar artificial tissue constructs of the presentinvention comprise a confluent endothelium and epithelium. Theepithelium comprises both type I and type II epithelial cells. Vesicleformation and cycling also occur in the constructs.

We have demonstrated that the type II cells are pseudoantigen-presenting cells. The artificial tissue constructs mountimmunological responses to bacterial stimulants and hormonalimmunosuppressants. The functioning of the artificial tissue constructwas also assessed by cytochrome P450 activity. Collectively, these datademonstrate that the tissue-engineered in vitro artificial tissueconstructs of the present invention are functional.

In one embodiment the present invention includes an artificial tissueconstruct comprising: (a) a first cellular layer comprising alveolarprimary epithelial cells and having a first face and a second face, and(b) a second cellular layer comprising alveolar primary endothelialcells, wherein the second layer is positioned on the first face or thesecond face of the first layer.

In preferred versions of this embodiment the alveolar primary epithelialcells are human cells, or the alveolar primary endothelial cells arehuman cells, or both the alveolar primary epithelial cells and thealveolar primary endothelial cells are human cells.

In versions of this embodiment where both the alveolar primaryepithelial cells and the alveolar primary endothelial cells are humancells, both the alveolar primary epithelial cells and the alveolarprimary endothelial cells are from the same human.

In other preferred versions of this embodiment the artificial tissueconstruct further comprise primary alveolar macrophages, preferablywherein the primary alveolar macrophages are interspersed among thealveolar primary epithelial cells and/or the alveolar primaryendothelial cells. The primary alveolar macrophages may also beinterspersed among the alveolar primary epithelial cells and thealveolar primary endothelial cells, and be positioned between the firstcellular layer and the second cellular layer. The artificial tissueconstruct may also comprise blood cells, preferably white blood cells.

In this embodiment of the invention a biocompatible membrane may bepositioned between the first cellular layer and the second cellularlayer of the artificial tissue construct. The biocompatible membrane maybe selected from the group consisting of basement membrane,extracellular matrix, collagen, laminin, proteoglycan, vitronectin,fibronectin, poly-D-lysine and polysaccharide. In a preferred embodimentthe biocompatible membrane is an extracellular matrix comprising lamininand collagen. In equally preferred embodiments the first cellular layerand the second cellular layer are in direct contact.

In a second embodiment the present invention is directed to methods ofdetermining whether a test compound has an immunological activity,wherein the method comprises:

(a) culturing an artificial tissue construct of the present inventionwith a test compound,

(b) assaying an immunological activity of the artificial tissueconstruct of (a), and

(c) comparing the immunological activity assayed in (b) to theimmunological activity assayed for the artificial tissue construct ofclaim 1 cultured in the absence of the test compound, wherein when thereis a difference in the immunological activity of the artificial tissueconstruct in the presence of the test compound compared to an absence ofthe test compound, the test compound is determined to have animmunological activity.

In this embodiment, the immunological activity may be selected from thegroup consisting of immunoglobulin generation, chemokine generation, andcytokine generation.

Also in this embodiment, the test compound may be selected from thegroup consisting of cigarette smoke, cigarette smoke particulates, anaerosol, a therapeutic agent, a biological compound, a vaccine, arespiratory bacterial disease pathogen, a respiratory disease viralpathogen, an environmental irritant, a diesel particle, a cosmeticingredient and a polycyclic hydrocarbon.

In an embodiment where the test compound is a vaccine, the vaccine maybe an influenza vaccine or a measles vaccine.

In an embodiment where the test compound is a respiratory diseasebacterial pathogen, the respiratory disease bacterial pathogen may beselected from the group consisting of Y. pestis, Bacillus anthracis, E.coli, Francisella tularensis, S. aureus, Group A beta-hemolyticstreptococci (GABHS), group C beta-hemolytic streptococci,Corynebacterium diphtheriae, Neisseria gonorrhoeae, Arcanobacteriumhaemolyticum, Chlamydia pneumoniae, Mycoplasma pneumoniae, Streptococcuspneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Bordetellapertussis, and B. parapertussis.

In an embodiment where the test compound is a respiratory disease viralpathogen, the respiratory disease viral pathogen may be selected fromthe group consisting of common flu, influenza A, influenza B,respiratory syncytial virus (RSV), adenovirus, parainfluenza virus,human parainfluenza virus (HPIV), metapneumovirus (hMPV), rhinovirus,coronavirus, coxsackievirus and herpes simplex virus.

In an embodiment where the test compound is a therapeutic agent, thetherapeutic agent may be an immunosuppressive drug or a small drugmolecule.

In an embodiment where the test compound is an immunosuppressive drug,the immunosuppressive drug may be a corticosteroid.

In an embodiment where the test compound is an immunosuppressivecorticosteroid hormone, the immunosuppressive corticosteroid hormone maybe selected from the group consisting of alclometasone, amcinonide,beclometasone, betamethasone, budesonide, ciclesonide, clobetasol,clobetasone, clocortolone, cloprednol, cortivazol, deflazacort,deoxycorticosterone, desonide, desoximetasone, dexamethasone,diflorasone, diflucortolone, difluprednate, fluclorolone,fludrocortisone, fludroxycortide, flumetasone, flunisolide, fluocinoloneacetonide, fluocinonide, fluocortin, fluocortolone, fluorometholone,fluperolone, fluprednidene, fluticasone, formocortal, halcinonide,halometasone, hydrocortisone, hydrocortisone aceponate, hydrocortisonebuteprate, hydrocortisone butyrate, loteprednol, medrysone,meprednisone, methylprednisolone, methylprednisolone aceponate,mometasone furoate, paramethasone, prednicarbate, prednisone,prednisolone, prednylidene, rimexolone, tixocortol, triamcinolone, andulobetasol.

In an embodiment where the test compound is an environmental irritant,the environmental irritant may be an environmental chemical irritant.

In an embodiment where the test compound is a polycyclic hydrocarbon,the polycyclic hydrocarbon may be 3-methylcholantherene orβ-nitrophenol.

In an embodiment where the test compound is a biological compound, thebiological compound may be selected from the group consisting of apeptide, a polypeptide, an antibody, a monoclonal antibody, anoligonucleic acid molecule, a polynucleic acid molecule, a saccharide,and a polysaccharide.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 show immunohistochemical staining of alveolar mucosal constructs.Panel a) is a bilayer structure showing a distinctive epithelium (AEPi)on top of endothelium (AEC). Panel b) shows an epithelium. Panel c)shows an endothelium. In each panel P=the porous Transwell surface.

FIG. 2 shows transmission electron microscopy images of epithelialcells. Panel a) shows type II (

) and type I (

) epithelial cells. Panel b) shows type II epithelial cells releasingsurfactant carrying vesicles (

).

FIG. 3 shows FM-143FX vesicle cycling in a bilayer alveolar mucosalconstruct. Panel a) is a confocal microscopy image showing fluorescentvesicles (V). Panel b) is a superimposed confocal and light microscopyimage showing cells and vesicles.

FIG. 4 shows an antigen uptake experiment with lung alveolar mucosalbilayer constructs in the upper row of panels, HLA-DR expression in themiddle row of panels, and co-localization of signals in the bottom row.Column a) is KLH. Column b) is TT.

FIG. 5 shows Alexa fluor 488™-labeled bacterial transport (24-hexposure) in E. coli-treated endothelium and epithelium bilayer (Panela); 20×) and S. aureus-treated endothelium and epithelium bilayer (Panelb); 10×).

FIGS. 6A-6B are a graphical representation of the effects of bacterialexposure on cytokine production in the bilayer construct. Whitebars=control; gray bars ═S. aureus; black bars=E. coli. FIG. 6A showsproduction levels of IL-6, IL-8, MCP-1, RANTES, and IP-10. FIG. 6B showsproduction levels of IL-12 (p40), IL-15, GM-CSF, IFN-γ, eotaxin andMIP-1α

FIGS. 7A-7B are a graphical representation of the effects of hormonaltreatment on cytokine production by the bilayer construct. Whitebars=control; gray bars=hydrocortisone; black bars=dexamethasone. FIG.7A shows production levels of IL-6, IL-8, MCP-1, RANTES, and IP-10. FIG.7B shows production levels of IL-12 (p40), IL-15, GM-CSF, IFNγ, Eotaxinand MIP-α.

FIG. 8 is a graphical representation of the cytochrome p450 activity ofthe bilayer mucosal construct. Induction agent: 3-methylcholantherine(3-MC), β-napthophenol (BNF). Detection Assay: 7-ethoxy resorufin(7-ERF), 7-pentoxy resorufin (7-PRF).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to artificial tissue constructscomprising two layers of cells, wherein one layer of cells is positionedatop the other layer of cells, and wherein one of the layers of cellscomprises alveolar primary epithelial cells and the other layer of cellscomprising alveolar primary endothelial cells. The present invention isalso directed to methods of determining whether a test compound hasimmunological activity, comprising culturing an artificial tissueconstruct of the present invention in the presence of a test compound,and determining the effect the test compound has on an immunologicalactivity of the artificial tissue construct. Immunological activities ofthe artificial tissue construct include immunoglobulin generation,chemokine generation and cytokine generation. The present inventionfurther provides methods for preparing the artificial tissue constructsof the present invention.

Artificial Tissue Constructs

The artificial tissue constructs of the present invention are tissueconstructs comprised primary of cells derived from respiratory tissuethat may be used to test and screen compounds in vitro for the potentialeffects the compounds might have on immunological activities of lungtissues in vivo.

At the most basic level, the artificial tissue constructs of the presentinvention comprise two layers of cells (cellular layers), wherein onelayer of cells is on top of and in contact with the other layer ofcells, for example, in the form of two coins that are stacked, one atopthe other. One of the layers of cells (at times referred to herein as a“first cellular layer” for sake of convenience and not to denote anylimitations regarding the placement of the layer in the constructs ofthe present invention) is comprised primarily of pulmonary alveolarrespiratory epithelial cells, preferably obtained from the alveolarregions of lung tissue. The pulmonary alveolar respiratory epithelialcells may be Type I cells, Type II cells or a mixture of Type I cellsand Type II cells. The skilled artisan will understand that such cellsare the epithelial cells of the alveoli that are in direct contact withair that enters the lungs of a mammal. The skilled artisan willunderstand that based on the particular use to which the artificialtissue constructs will be put, other types of respiratory epithelialcells may be used.

The second of the layers of cells (at times referred herein to as a“second cellular layer” for sake of convenience and not to denote anylimitations regarding the placement of the layer in the constructs ofthe present invention) is comprised primarily of respiratory endothelialcells, preferably vascular endothelial cells obtained from the alveolarregions of lung tissue. The skilled artisan will understand that suchcells are the endothelial cells of capillaries in contact with thealveoli that are in direct contact with the blood of the capillaries.Thus, the artificial tissue constructs of the present invention mimic instructure the two cell layers that comprise mammalian alveoli.

The cells that make up the cellular layers are mammalian cells,including human, simian, mouse, rat, rabbit, guinea pig, horse, cow,sheep, goat, pig, dog and cat cells. Preferably the cells are humancells. The cells that comprise the constructs may come from a singleindividual mammal, such a one man, or come from more than oneindividual. When from more than one individual the cellular layers maybe each comprises of cells from a different individual, or the cellscomprising each layer may be from different individuals.

The cells that make up the layers may be obtained from existing cellcultures, or produced de novo by disassociating primary epithelial andendothelial cells from lung tissue and culturing such cells underappropriate conditions to form the cellular layers.

The artificial tissue constructs of the present invention also maycomprise a biocompatible membrane that is positioned between the twolayers of cells. Alternatively, both cellular layers may be positionedon top of the biocompatible membrane. Such biocompatible membranes mayserve to maintain the layers of cells as discrete cellular layers in thecontrast. The biocompatible membrane may also provide mechanical supportfor the constructions. The biocompatible membrane further provide ameans for allowing selective passage between the two cellular layers andmay thus be impermeable, semi-permeable or porous, thereby controllingthe ability of fluids, gases, small molecules and cells to pass from onecellular layer to another. The biocompatible membrane may be anymaterial that does not have a deleterious effect on the basic nature ofthe artificial tissue construct. Suitable examples of biocompatiblemembranes include materials comprising basement membrane, extracellularmatrix, collagen, laminin, proteoglycans, vitronectin, fibronectin,poly-D-lysine and/or polysaccharides. When the biocompatible membranecomprise extracellular matrix it may be comprised of laminin andcollagen.

The thickness of the biocompatible membrane will differ depending on thematerial that is being used and the composition of the cellular layersin the constructs. However, it is envisioned that the biocompatiblemembrane will range in size from less than about 200 nm to more thanabout 1 mm. In preferred embodiments the biocompatible membrane is about0.5 nm to about 1 um.

The skilled artisan will understand that the constructs of the presentinvention may also comprise constructs where the first cellular layerand the second cellular layer are in direct contact.

The artificial tissue constructs of the present invention may be ofvarying sizes and shapes, and the skilled artisan will understand thatthere are few limitations on the three-dimensional structure of theconstructs. Thus, the cellular layers may be of any shape, includinground, oval, rectangular, triangular and square. The cellular layers mayeach of be a different shape or size, or of the same general shape orsize. The cellular layers may be of different thicknesses, or have thesame general thickness. It is convenient to consider each cellular layeras being a very flat structure, resembling the structure of a coin, thathas a first face and a second face in opposition to each other (the“heads” and “tails” sides of a coin), as well as an narrow edge thatencompasses the circumference of the layer. In preferred embodiments thetwo layers are position such that the face of one layer is positioned inparallel and against the face of the other layer, such as a stack of twocoins mentioned above. However, as also indicated above the two layersmay be in direct contact or separated by the biocompatible membrane. Thetwo layers may be positioned entirely against each other or may bepositioned with less than the entire layers being position against eachother. The first cellular layer may be positioned on top of the secondcellular layer, or the second cellular layer may be positioned on top ofthe first cellular layer.

The artificial tissue constructs of the present invention may beprepared, grown and maintained in any suitable tissue culture vesselthat permits production, growth and maintenance of the constructs.Suitable vessels include Transwell™ permeable support devices and T-75flasks.

The artificial tissue constructs of the present invention may beprepared by, for example, from autologous epithelial and endothelialcells that are isolated from normal human tissue biopsy samples byliterature procedures. A tissue culture vessel (plate or flask, e.g.,Transwell™ buckets or a T-75 flask) may be coated with fibronectin (5-50mg/mL) and placed in an incubator at 37° C. prior to seeding with cells.Endothelial cells can be disassociated from lung tissue samples andadded to the tissue culture vessel, for example, at a density of˜25,000-35,000 cells/well in a Transwell™ bucket. The cells can becultured until approximate confluency is reached. Endothelial cells canthen be disassociated from lung tissue samples and added to the tissueculture vessel containing the confluent endothelial cells, with seedingat a density of ˜20,000-25,000 per well. The epithelial cells can becultures in, for example, Media 199 (M199) and Dulbecco's minimumessential medium (DMEM). The resulting artificial tissue constructs ofthe present invention may be maintained by culturing them at 37° C. in5% CO₂.

The artificial tissue constructs of the present invention may alsocomprise additional cell types in addition to the alveolar epithelialand vascular endothelial cells described above. For example, theconstructs may further comprise primary alveolar macrophages. Theprimary alveolar macrophages may be interspersed with the first cellularlayer, the second cellular layer, or both. The primary alveolarmacrophages may also be within the biocompatible membrane. The skilledartisan will understand that primary alveolar macrophages will migratewithin and to different regions of the construct, and within and betweenthe cellular layers. The artificial tissue constructs may also containone or more of the following cell types: red blood cells, white bloodcells, including monocytes, T lymphocytes (including CD4+ and CD8+ Tcells), B lymphocytes, and natural killer cells,

The artificial tissue constructs of the present invention may alsocomprise additional components, including cytokines and growth factors.

The present invention is also directed to methods of determining whethera test compound has immunological activity, comprising culturing anartificial tissue construct of the present invention in the presence ofa test compound, and determining the effect the test compound has on animmunological activity of the artificial tissue construct.

The test compounds assayed in these methods are any for which one wishesto determine the effect the compound has on an immunological activity ofthe lung of a mammal. It will be readily apparent to the skilled artisanthat the test compounds will include those compounds which are suspectedof causing a deleterious effect on lung tissue, such environmentalpollutants and irritants, such as emissions and particulates containedtherein from various manufacturing processes and facilities, powergenerating facilities and internal combustion engines, allergens;cigarette smoke and the particulates contained within cigarette smoke(such as nicotine, hydroquinone, and benzopyrene); a cosmeticingredient; a polycyclic hydrocarbon (such as 3-methylcholantherene andβ-nitrophenol); respiratory bacterial disease pathogens; and respiratorydisease viral pathogens.

Allergens include antigenic compounds that may be inhaled, such asmolds, ragweed, tree and grass pollens, pet dander, and house dustmites.

Respiratory disease bacterial pathogens include Y. pestis, Bacillusanthracis, E. coli, Francisella tularensis, S. aureus, Group Abeta-hemolytic streptococci (GABHS), group C beta-hemolyticstreptococci, Corynebacterium diphtheriae, Neisseria gonorrhoeae,Arcanobacterium haemolyticum, Chlamydia pneumoniae, Mycoplasmapneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, Moraxellacatarrhalis, Bordetella pertussis, B. parapertussis, M. pneumoniae andC. pneumoniae. Respiratory disease viral pathogens include common flu,influenza A, influenza B, respiratory syncytial virus (RSV), adenovirus,parainfluenza virus, human parainfluenza virus (HPIV), metapneumovirus(hMPV), rhinovirus, coronavirus, coxsackievirus and herpes simplexvirus.

Test compounds include compounds of a medical nature that might be usedin the prevention and/or treatment of disease. Such compounds includeaerosols and the components thereof that might be used as a carrier inthe administration of a therapeutic agent, vaccine or other compound toa subject via the lungs, vaccines themselves (such as an influenzavaccine or a measles vaccine), therapeutic agents themselves (such as asmall drug molecule or an immunosuppressive drug, e.g., animmunosuppressive corticosteroid hormone), and other biologicalcompounds (such as a monoclonal antibody, a peptide, a polypeptide, anoligonucleic acid molecule, a polynucleic acid molecule, a saccharide,and a polysaccharide).

Immunosuppressive corticosteroid hormones include alclometasone,amcinonide, beclometasone, betamethasone, budesonide, ciclesonide,clobetasol, clobetasone, clocortolone, cloprednol, cortivazol,deflazacort, deoxycorticosterone, desonide, desoximetasone,dexamethasone, diflorasone, diflucortolone, difluprednate, fluclorolone,fludrocortisone, fludroxycortide, flumetasone, flunisolide, fluocinoloneacetonide, fluocinonide, fluocortin, fluocortolone, fluorometholone,fluperolone, fluprednidene, fluticasone, formocortal, halcinonide,halometasone, hydrocortisone, hydrocortisone aceponate, hydrocortisonebuteprate, hydrocortisone butyrate, loteprednol, medrysone,meprednisone, methylprednisolone, methylprednisolone aceponate,mometasone furoate, paramethasone, prednicarbate, prednisone,prednisolone, prednylidene, rimexolone, tixocortol, triamcinolone, andulobetasol.

The immunological activities for which the test compounds may be assayedinclude all of the immunological activities that take place in the lungsof a mammal. Examples of immunological activity include up-regulation ofhost defense genes (including MUC2, MUC5C, hBD2, and LL37/CAP18),immunoglobulin generation, chemokine generation (such asinterferon-inducible T-cell α-chemoattractant (I-TAC), andγ-interferon-inducible T cell chemoattractant), and cytokine generation(including interleukins (IL-1, IL-5, IL-6, IL-8), RANTES (regulated uponactivation, normal T cell-expressed, and secreted), endothelin,granulocyte-monocyte colony stimulating factor (GM-CSF), transforminggrowth factor beta (TGF-β) and interferon-γ-induced protein (IP-10).

The effect a test compound has on a particular immunological activity ofthe constructs of the present invention will be determined in a somewhatdifferent manner depending in the nature of the test compound, thecomposition of the artificial tissue construct and the particularimmunological activity being assayed. However, the general method willbe the same regardless of these variables and includes:

(a) culturing the artificial tissue construct with a test compound,

(b) assaying a selected immunological activity of the artificial tissueconstruct, and

(c) comparing the values determined in the assay to the values of thesame assay performed using an artificial tissue construct with the samecomposition as in (a) but cultured in the absence of the test compound(or in the presence of a control). The difference between the valuesdetermined for the assay in the presence and absence of the testcompound will provide specific information regarding the effect the testcompound has on the immunological activity of the artificial tissueconstruct, which may be extrapolated to the potential effect the testcompound will have on the immunological activity of the lung and lungtissue of a living mammal.

As indicated above, the particular assay used in the methods of thepresent invention will vary, primarily based on the particularimmunological activity being assayed. For example, when theimmunological activity being assayed is up-regulation of host defensegenes, one might assay for RNA production (via northern blot analysis)or protein production (via a western blot analysis) corresponding tothose genes known to be up-regulated in a host defense reaction.Similarly, where the immunological activity being assayed isimmunoglobulin generation one might assay for immunoglobulin RNAproduction (via northern blot analysis) or protein production (via awestern blot analysis).

Where the immunological activity being assayed is chemokine or cytokinegeneration, one might again assay for RNA production (via northern blotanalysis) or protein production (via a western blot analysis)corresponding to particular chemokines or cytokines.

In the examples that follow, immunohistochemical staining of the bilayerconstructs demonstrate the endothelial/epithelial bilayer structure ofthe artificial tissue constructs. The functionality of the epitheliumwas shown by vesicle cycling analysis of FM-143FX, tetanus toxoid andkeyhole limpet hemocyanin across the mucosal construct. Experimentaldata generated with an artificial tissue construct of the presentinvention showed that it responded to chemical stimuli, such asimmunosuppressant hormones (e.g., dexamethasone, hydrocortisone) andbacterial treatments (e.g., S. aureus, E. coli). Cytochrome P450activity experiments also showed that the alveolar cells had significantP450 activity that can be quantified after induction by variousirritants.

EXAMPLES Example 1 Fabrication of Bilayer Artificial Tissue Constructs

In an embodiment of the present invention, artificial tissue constructswere prepared by creating a bilayer cellular structure on top of acollagen and laminin-coated porous plastic membrane. Primary cellisolations from normal human tissue biopsy samples were performed toisolate autologous epithelial and endothelial cells. The tissue wasstored in preservation media for up to ˜24 h before cell isolation. Thecell isolation protocol was similar to that in previous reports (Bur etal. (2006) Eur. J. Pharm. Sci. 28, 196-203; Elbert et al. (1999) Pharm.Res. 16, 601-8). Enzyme-based tissue digestion was performed withelastase and trypsin, followed by IgG panning to isolate different cellpopulations.

Experiments were performed with a series of extracellular matrixproteins and chitosan, a natural polysaccharide, as examples.Specifically, collagen, fibronectin, laminin, Matrigel™, collagen, andchitosan-blended extracellular matrix proteins were examined. Blendedand cross-linked materials were cased into gels, porous scaffolds, andcoatings to determine the effects of material microstructure on tissuegeneration in vitro. The gelled materials were prepared with collagenand collagen-blended fibronectin or laminin by controlling the pH of theprotein solutions with buffers at controlled temperatures until thepolymers formed a physical gel, based charge density. The porousscaffolds were prepared using chitosan-based materials with or withoutcollagen and laminin. The porosity of the scaffold was controlled byadjusting the density of the polymer solutions, freezing the solutionunder controlled temperature conditions, and later lyophilizing themixture.

In a preferred embodiment, the coatings were prepared by pouring thepolymer solutions (collagen, including type 4 collagen, and laminin) onthe porous support. The thin polymer coating was then aspirated off thesupport and the coated surfaces were allowed to air dry prior to cellseeding. Primary alveolar endothelial cells were cultured on top of theplastic membranes until they reached confluency. The alveolar epithelialcells were then seeded on top of the endothelial cells, to create anepithelium.

In a preferred embodiment of the present invention, the followingprotocol was used to generate artificial tissue constructs. Suitablematerials can be purchased, for example, from the following suppliers:penicillin-streptomycin-glutamine (Sigma), Media199 (Invitrogen),Dulbecco's minimum essential medium (Invitrogen), fibronectin (BDBioscienes), ethanol (Sigma), collagen (Inamed), laminin (BDBiosciences), HTS-Transwell™ plates (Corning Life Sciences),trypsin-EDTA (Sigma), 1×PBS (Sigma), fetal bovine serum (Hyclone), humanalveolar endothelial cells (ScienCell), and human alveolar epithelialcells (ScienCell).

For the endothelial cell culture, endothelial cell media was used. Media199 (M199) stock solution containing 1%penicillin-streptomycin-glutamine was prepared, and was supplementedwith 5-15% (v/v) fetal bovine serum. A tissue culture vessel (plate orflask) was coated with fibronectin (5-50 mg/mL) and placed in theincubator for ˜1 h. Later, the fibronectin was removed andserum-containing media was added to the tissue culture vessel. Forexample a volume of 12 mL of 10% serum containing culture media wasadded to a T-75 flask coated with 5 mg/mL of fibronectin solution. Avial of human alveolar endothelial cells was thawed at 37° C. and thecell suspension was added to the coated tissue culture vessel. The flaskwas placed in the incubator and the media was changed ˜24 h after cellseeding. The media was changed every ˜48 h after the initial ˜24-hincubation period until the cells reached ˜90% confluency.

To subculture the cells, another fibronectin-coated vessel was prepared.PBS, tissue dissociation enzyme (like trypsin-EDTA), andserum-containing media were warmed to 37° C. The cells were rinsed withPBS and trypsin-EDTA solution was added. The detached cells (roundmorphology) were observed under a light microscope. Media containing5-15% serum was then added to the cell suspension and then centrifuged(˜1000 rpm, ˜10 min) The supernatant was aspirated and the cell pelletwas re-suspended in serum-containing media. The cells were counted anddiluted as needed. The cells were then seeded in tissue culture vessels.For example, a seeding density of ˜20,000-35,000 cells/well was used forthe multi-well Transwell™ devices (0.33 cm²).

For the epithelial cell culture, epithelial cell media was used. Media199 (M199) and Dulbecco's minimum essential medium (DMEM) stocksolutions containing 1% penicillin-streptomycin-glutamine were prepared.A 5-15% (v/v) serum-containing culture media was prepared in 50:50solution of DMEM and M199. The tissue culture vessel was coated withfibronectin (5-50 mg/mL) and placed in an incubator (37° C.). Afterapproximately 1 h, serum-containing tissue culture media was then addedto the coated tissue culture vessel. For example, a volume of 12 mL of10% serum containing culture media was added to a T-75 flask coated with5 mg/mL of fibronectin solution. A vial of human alveolar epithelialcells was thawed at 37° C. and the cell suspension was added to theserum-containing media. The cells were cultured in an incubator andmedia was changed ˜24 h after cell seeding. The media was changed every˜48 h after the initial ˜24-h incubation period until the cells reached˜90% confluency.

To subculture the cells, another fibronectin-coated vessel was prepared,as described above. PBS, tissue dissociation enzyme (like trypsin-EDTA),and serum-containing media were warmed to 37° C. The cells were rinsedwith PBS and Trypsin-EDTA solution was added. The detached cells (roundmorphology) were observed under a light microscope. 5-15%serum-containing media was then added to the cell suspension and thencentrifuged (˜1000 rpm, ˜10 min) The supernatant was aspirated and thecell pellet was re-suspended in serum-containing media. The cells werecounted and diluted as needed. The cells were then seeded in tissueculture vessels. For example, a seeding density of ˜25,000-35,000cells/well was used for the multi-well Transwell™ devices (0.33 cm²).

In a preferred embodiment of the present invention, a mucosal constructwas prepared on multi-well HTS-Transwell™ plates. The cell growthsurface area of a Transwell™ bucket is 0.33 cm². The coating volumes andcell densities can be scaled, based on the cell growth surface area ofthe vessel or surface of interest.

HTS-Transwell™ plates (24-well) were coated with 0.1-1% (w/w) lamininand collagen solution: the dilutions were prepared in M199/DMEM. Theplates were incubated at room temperature for ˜1 h. The collagen/lamininsolution was then aspirated off and ˜75 μL of media was added to thecoated membrane: ˜300 μL of media was added to the bottom well. Theplates were placed in the incubator overnight.

Human alveolar endothelial cells were added to the Transwell™ buckets,at a density of ˜25,000-35,000 cells/well. The media was changed ˜24 hafter cell seeding and at ˜48-h intervals for one week, by when theendothelial cells reached approximate confluency. Human alveolarepithelial cells were then added on top of the endothelial cells at aseeding density of ˜20,000-25,000 per well.

In another embodiment, if a higher density of Type II alveolarepithelial cells was desired, then the confluent endothelial cell layerwas coated with ˜50 μL collagen-laminin solutions, as described above.The coating solution was then aspirated, and the cells were incubatedwith ˜75 μL of serum-containing epithelial cell media for ˜30 min Themedia was then aspirated and epithelial cells were added on top of thecoated endothelial cell layer, at a density of ˜20,000-30,000 cells perwell. The media was changed ˜24 h after cell seeding and at ˜48-hintervals for one week, by which time the endothelial cells had reachedapproximate confluency. It usually took ˜2 to ˜3 days for the epithelialcells to grow to approximate confluency.

Example 2 Characterization of the Constructs

Immunohistochemical staining of the mucosal construct was used toidentify the presence and location of the epithelial and endothelialcells in the bilayer structure. Epithelial cells in the construct wereidentified by immunohistochemical staining of a number of alveolarcell-specific cytokeratins, including CK-1, 4, 5, 6, 8, 10, 13, 18, 19.

In the staining method formalin-fixed tissue was embedded in paraffinwax and sectioned at 5 μm. The paraffin wax was then dissolved withxylene and sections were blocked with ˜10% goat serum. A primarycytokeratin antibody cocktail was then added, followed by the additionof the biotinylated secondary antibody. Streptavidin-peroxidase enzymeconjugate was then applied and aminoethyl carbazole (AEC) chromagen waslater added to the sections. The samples were counterstained withhematoxylin and rinsed thoroughly with dH₂O and incubated until colordeveloped. Light microscopy was used to image the cytokeratin-containingepithelial cells in the in vitro-grown mucosal constructs.

Alveolar endothelial cells in the mucosal construct were identified byFactor VIII staining, specific for endothelial cells. The formalin-fixedtissue samples were paraffin wax-embedded and sectioned at 5 μm forantibody staining. The tissue was then deparaffinized and blocked with10% goat serum to reduce non-specific staining. Primary antibody wasapplied for 1 h at room temperature or incubated overnight at 4-6° C.,followed by a biotinylated secondary antibody for 30 min. Astreptavidin-peroxidase enzyme conjugate was then applied and aminoethylcarbazole (AEC) chromagen was later added. Light microscopy of thesections showed the red staining of Factor VIII on the endothelialcells. The tissue integrity of each cell monolayer was characterized bytight junction staining of ZO-1, E-cadherin, and occludin. Confocalmicroscopy was performed on the formalin-fixed epithelial andendothelial tissue.

Double staining of both alveolar epithelial and endothelia cells showeda clear bilayer structure of the mucosal construct on top of a porousplastic support (FIG. 1). The top layer of cells showed cytokeratinstaining, characteristic of epithelial cells and the bottom cell layershowed Factor VIII staining, characteristic of endothelial cells.Individual staining of the epithelium and endothelium also showed asingle layer of tissue per cell type. Tight junction staining of theendothelium and the epithelium showed a confluent cell layer.

Example 3 Type II Alveolar Epithelial Cell

After successfully expanding and co-culturing human epithelial andendothelial cells on laminin-coated membranes and porous scaffolds, cellmorphology and activity was examined. In particular, the presence ofType II alveolar epithelial cells was studied. These cells participatein vivo in primary inflammatory and immune reactions in lung mucosatowards external contaminants and pathogens. Type II cells produce alung surfactant substance that comprises lipoproteins and surfactantproteins able to bind polysaccharides of bacterial membranes, andenhance protective release of oxygen radicals by alveolar macrophages.

The morphology of Type I and Type II epithelial cells was examined bytransmission electron microscopy of epoxy-embedded andmicrotome-sectioned samples of the mucosal tissue constructs of thepresent invention. Briefly, cultured epithelial cells were fixed with2.5% gluteraldehyde and were later exposed to osmium tetroxide. Serialethanol dehydration was then performed, followed by tissue infiltrationof propylene oxide. The samples were then transitioned through propyleneoxide and epoxy resin, followed by embedding in 100% epoxy resin. Epon812 along with dodenyl succinic anhydride (DDSA), nadic methyl anhydride(NMA), and 2,4,6-tri(dimethylaminomethyl) phenol (DMP-30) were used toprepare the embedding polymer resin. The samples were sectioned at 100nm using a Leica UltraCut™ Microtome. Sections were then stained with 2%uranyl acetate followed by a lead citrate treatment. The TEM used was aHitachi H-7000™.

Visualization of Type II cells with the FM143-FX fluorescent dye wasbased on exocytotic activity of the Type II cells. The amphiphilicfluorescent styryl dye FM143-FX, or(N-(3-triethylammoniumpropyl)-4-[dibutylaminostyryl) propidiniumdibromide) is actually non-fluorescent in an aqueous environment, butbecomes fluorescent when it comes in contact with lipid-containingplasma membranes and vesicles. Type II alveolar cells release vesiclesfilled with surfactant proteins and lipoproteins through fusion pores.Infiltration of the FM143-FX dye through these pores into thephospholipid-containing lamellar bodies (pre-formed vesicles) and plasmamembranes results in bright red fluorescence. Two-week old constructscontaining either alveolar epithelial or endothelial cells or acombination of both cell types were used for the cell transport studies.Tissue constructs were fixed with 4% paraformaldehyde and 5 μg/mL ofFM143FX (Invitrogen) in HEPES-containing media was added to the treatedconstructs. Cells were visualized with an Olympus Fluoview FV300™confocal laser scanning biological microscope. The mucosal tissue wasvisualized at various depths to locate the fluorescentFM143FX-containing vesicles.

Staining of epithelial cytokeratins was performed on formalin-fixed,non-embedded, non-sectioned epithelial/endothelial constructs, using ananti-pan cytokeratin immunohistochemical cocktail (Sigma-Aldrich) thatcontains monoclonal antibodies to human CK-1, 4, 5, 6, 8, 10, 13, 18,19, that are alveolar epithelial tissue-specific. Apart from the primarymonoclonal antibody, all reagents were from Invitrogen.

The tissue construct was fixed in neutral buffered formalin containing˜10% goat serum was used as a blocking solution. The primary antibodypan-cytokeratin cocktail was then added and samples were incubated atroom temperature. Biotinylated secondary antibody was then addedfollowed by a repeated incubation period. Streptavidin-peroxidase enzymeconjugate was then applied and aminoethyl carbazole (AEC) chromagen waslater added to the constructs. The samples were then counterstained withhematoxylin and rinsed thoroughly with dH₂O and incubated until colordeveloped. Light microscopy was used to image the cytokeratin containingepithelial cells (purple) in the in vitro-grown mucosal constructs.

TE images of Type I and Type II cells revealed multiple lamellar bodiesand vesicles filled with surfactant, which helped to discriminate Type Iand Type II cells. These results show that the collagen- andlaminin-containing scaffold materials did promote the growth Type IIalveolar epithelial cells in culture. Type II cells are important intransport/immunological processes across alveolar surfaces.

Example 4 Evaluation of Tissue Function: Vesicle Cycling, and AntigenUptake and Presentation

Primary tissue functionality was tested by checking for vesicle cyclingability and antigen uptake and presentation by the artificial tissueconstructs, specifically the Type II epithelial cells. The presence ofType II cells in the epithelium is important for actively bringingantigens across the mucosal barrier. Several techniques were used toidentify the cell populations and tissue architecture of the artificialtissue constructs. The presence of Type II epithelial cells is importantin a functional alveolar artificial tissue construct, because thesecells are primarily responsible for the foreign body response. Both TypeI and Type II epithelial cells were identified in the artificial tissueconstructs of the present invention. The Type II cells also releasesurfactant-carrying vesicles. We also demonstrated the immunologicalfunction of the type II cells in the bilayer constructs of the presentinvention.

Transmission electron microscopy (TEM) and immunohistochemical stainingof the artificial tissue construct were used to identify Type II cells.Briefly, the cultured epithelial cell cultures were fixed withgluteraldehyde and later exposed to osmium tetroxide. The samples werethen embedded in Epon resin, sectioned, and stained with uranyl acetateand lead citrate (FIG. 2). Immunohistochemical staining of the mucosalbilayer was used to identify the epithelium and endothelial monolayers.Epithelial cells in the construct were identified alveolar cell-specificcytokeratin (CK-1, 4, 5, 6, 8, 10, 13, 18, 19) staining. Lightmicroscopy was used to image the cytokeratin-containing epithelial cellsmucosal constructs.

The alveolar endothelial cells in the bilayer mucosal construct wereidentified by Factor VIII staining and light microscopy of the sectionedbilayer tissue. Tissue integrity was visualized by tight junctionstaining of ZO-1, e-cadherin, and occludins (data not shown).

Example 5 Vesicle Cycling in Epithelial Cells

Confocal microscopy of the constructs was performed at various depthsfrom the surface of the cells (FIG. 2). The total time for vesiclecycling was fixed to ˜10 min after the addition of the dye. The resultsshow the presence of an epithelium of approximate 10-μm thickness. Nosignificant level of fluorescence was detected for the endotheliummonolayer construct. For the bilayer constructs there is a ˜3 μm shiftfor the epithelium below the endothelium tissue. The ˜3-μm shift invesicle location in the epithelium over endothelium construct can beattributed to the initial cell seeding orientation of the activeepithelial cells.

The vesicle cycling activity of Type II cells was visualized withFM143-FX fluorescent dye (FIG. 2). It was shown that the alveolarepithelial cells in the construct were actively forming vesicles aroundthe FM143-FX molecules. However, a need to control the time dependencyof this process was identified, as the initial confocal microscopyimages showed the presence vesicles over a larger distance.

Example 6 Endpoint Kinetic Analysis of Vesicle Cycling

The endocytotic/exocytotic activity of Type II epithelial cells wasvisualized with FM-143X. This amphiphilic molecule is non-fluorescent insolution. The alveolar epithelial cells in the construct were indeedactively forming vesicles around the FM-143FX molecules. In thisexperiment the cells were exposed to FM-143FX for a short time. Confocalmicroscopy was performed on four different tissue motifs: monolayerepithelium, endothelium and bilayer structures containing epitheliumover endothelium, and vice versa. Clear vesicle formation around thefluorescent FM-143FX molecules was visualized in the epithelium over ashort time, indicating that the Type II cells in the epithelium werehighly active (FIG. 3).

The exact location of the epithelium with respect to the porous plasticsupport was unclear. In this experiment the cells were exposed to ˜5-10μg/mL of FM-143FX for a fixed period of time (10 min). Confocalmicroscopy was performed on four different tissue motifs: monolayerepithelium, endothelium and bilayer structures containing epitheliumover endothelium and vice versa.

Surfactant vesicles cycling across the mucosal construct were examined.Samples of pure endothelial TE, pure epithelial TE, and combinedepithelial/endothelial TE constructs were grown on laminin-coated24-well collagen Transwell™ plates. The constructs were stained withFM143-FX to study the activity of the epithelium. Confocal images wererecorded at various depths from above and below the surface of the celllayers. Bright fluorescent images were obtained for epithelial andcombined epithelial/endothelial constructs, while pure endothelialculture produced negligible fluorescence. The presence of red vacuoleswas indicative of active transport of the fluorescent dye by the Type IIepithelial cells. This result demonstrated a functional epithelium inthe in vitro-cultured constructs.

Example 7 di-I-acetylated LDL Endocytosis Method

The di-I-acetylated LDL endocytosis method was used to visualizeendothelial cells in the alveolar epithelial and endothelialcell-containing tissue constructs. The presence of red fluorescent dotsindicates the location of the fluorescent dye in the endothelial celllayer in the construct. The endothelial cells were cultured on top ofthe epithelial cells; thus, the dye is not visible in the layer below(the epithelium). Light and fluorescence microscopy superimposed imagesshow the endothelial cell location with respect to the epithelium.

Staining endothelial cells with Di-I-acetylated LDL was performed onnon-embedded, non-sectioned epithelial/endothelial constructs.Di-I-acetylated LDL, or1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanineperchlorate-labeled acetylated low-density lipoprotein entersendothelial cells via receptor-mediated endocytosis and eventuallyappears in lysosomes. The dye produces intense red fluorescence. 15μg/mL of DiI acetylated LDL (Invitrogen) was added to two-week-oldtissue constructs, containing both epithelial and endothelial cells.Cells were serum-deprived before the addition of DiI acetylated LDL.Samples were incubated for ˜3 h at ˜37° C. to permit endocytosis of thefluorescent probe. Cells were examined with an Olympus Fluoview FV300™confocal laser scanning IX-81 biological microscope. The tissue wasvisualized at various depths to locate the fluorescent DiI acetylatedLDL-containing vesicles.

These studies demonstrated the presence of a significant number of TypeII epithelial cells in the constructs of the present invention. Stainingwith amphiphilic fluorescent styryl dye FM143-FX and usingdepth-stratified confocal imaging confirmed intensive endo- andexocytosis performed by Type II epithelial cells. Staining withanti-pan-cytokeratin (epithelial-specific) and di-I-acetylated-LDL(endothelial-specific) revealed features of epithelial and endothelialcomponents of the combined constructs.

Example 8 Labeled Antigen Uptake by Alveolar Mucosal Cells and HLA-DRExpression

While respiratory mucosal dendritic cells (DCs) serve as primaryaccessory cells, the resident epithelial cells also function asantigen-presenting cells (APCs) in the upper and lower respiratorymucosa. They also express the MHC class II molecules needed for T cellactivation. The large surface area of the respiratory mucosa directlyexposes the lung epithelium to airborne antigens.

Antigen uptake of fluorescently labeled TT and KLH by the tissueengineered alveolar artificial tissue construct was allowed and laterthe tissue was stained for HLA-DR to visualize the co-localization ofthe engulfed antigen and the MHC class II expression by the cells. TTand KLH were labeled with fluorescein isothiocyanate (FITC; 1:5 molarratio) to create a stable thiourea bond. The labeled product wasisolated by membrane filters with appropriate molecular weight cut off.The cells were exposed to the labeled antigen for a period of one hour.The tissue bilayer was then washed with PBS and visualized with confocalmicroscopy. Anti-HLA-DR-APC-Cy7 was then added to visualize the presenceof MHC class II molecules. The tissue was imaged after half an hourafter the treatment and subsequent washings (FIG. 4). KLH and TT weresuccessfully labeled and processed by the alveolar artificial tissueconstructs where some vesicle-like structures were visualized. It can beseen that most of the HLA-DR signal is localized near the FITC-antigenfluorescence; however there is some low intensity signal by other cellsas well. The presence of yellow fluorescence shows co-localization ofthe captured antigen and HLA-DR expression indicating that the mucosalcells may be useful in presenting antigen to T cells.

Example 9 Effects of Environmental Irritants

Environmental irritants (most often referred to as allergens) are commonantigenic compounds that are inhaled, such as molds, ragweed, tree andgrass pollens, pet dander, and house dust mites. Grass and house dustmites are the most common environmental irritants, affecting over 22% ofthe population (Son & Endre (2005) Orv Hetil. 146, 833-7).

The effects of, for example, other environmental irritants, such asairborne polycyclic aromatic hydrocarbons, and cigarette smoke-relatedcarcinogens can be examined. Polycyclic hydrocarbons are aromaticorganic byproducts of industrial chemical and combustion processes.Respiratory epithelium responds to such compounds by cytochrome P450enzymatic activity and cytokine production.

The effects of carcinogens, such as 3-methylcholantherene andbeta-napthoflavone, and other airborne environmental irritants, such asdiesel particles and urban dust, that have been characterized by, forexample, NIST (standardized irritants), can be examined using the modelsof the present invention, as can the effects of cigarette smokecarcinogens, such as nicotine, hydroquinone, and benzopyrene. It hasbeen reported that animals exposed to particulate nicotine exhibit lossof antibody responses to T cell proliferation and an overallimmunosuppressive response; data suggests that after binding to nicotineT cells lose their ability to enter cell cycle and proliferate (Soporiet al. (1998) Adv. Exp. Med. Biol. 437, 279-89; Geng et al. (1996) J.Immunol. 156, 2384-9). Hydroquinine and benzopyrene also elicitimmunosuppressive responses due to T cell cycle interference (Rodriguezet al. (1999) Immunopharmacol. Immunotoxicol. 21, 379-96; Li et al.(1996) Toxicol. Appl. Pharmacol. 139, 317-23). BioPlex-based assays canbe used to monitor cytokine levels in culture media.

Example 10 Effects of Environmental Irritants and Hormones on Constructs

Immunosuppressant effects on the cultured mucosal construct were testedby the addition of, for example, dexamethasone (0.5-0.1 μM) andhydrocortisone (5-10 μM) for 24 h. The effect of, for example, dieselparticles (0.01-0.2 mg/mL) and urban dust (0.01-0.2 mg/mL) exposure wasalso studied in the alveolar tissue. NIST standards Diesel ParticulateMatter (SRM 2975), and Urban Dust (SRM 1649a), containing both organicand inorganic components, were used as environmental irritants (FIG. 3).A BioPlex 22-Plex™ cytokine kit (Upstate) was used to quantify thecytokine levels in the collected media.

The cytokine profile of the bilayer construct prepared in a 0.33 cm²area Transwell™ was compared with the alveolar epithelial cells culturedin T-25 flasks. Controls for each experiment with or without the variousirritant treatments were also included in the experimental design.

Example 11 Tissue Functionality Evaluation: Cytokine Production

The effects of immunosuppressant hormonal treatment (dexamethasone andhydrocortisone) and exposure to bacterial particles (E. coli andStaphylococcus aureus) on respiratory cells were examined by quantifyingcytokine production by the mucosal tissue. The cellular bilayer wasexposed to either high or low levels of hydrocortisone, dexamethasone,or E. coli or S. aureus particles for 24 h.

Cellular uptake of WST-1 was used to determine the mitochondrialactivity of live cells. A spectroscopic reading of thecollected/metabolized tetrazolium salt was performed to compare thecellular activity of hormone or bacterial particle treated cells to thenon-treated control cells after 24-h treatments. The alveolar artificialtissue construct cells were activated exposure to 3-methylcholantherewhile the cells were less activated with the BNF treatments. Theseresults indicated that the bilayer mucosal construct showed similarfunctionality to primary lung tissue.

Example 12 Bacterial Exposure

The open respiratory system is prone to pathogenic bacterial infections.As examples, E. coli (Gram-negative) and Staphylococcus aureus(Gram-positive) bacteria can use many substrates as vehicles for growthand transport into the lungs. Both bacteria carry numerous cell surfaceorganelles that help them enter mucosal surfaces, where they causerespiratory illnesses.

The ability of the mucosal constructs to endocytose heat-inactivated,fluorescently labeled bacterial particles was examined. The effects ofbacterial treatment on cytokine production by the mucosal models wereassessed. Such experiments were conducted with the alveolar artificialtissue construct. Similar experiments can be conducted with anasopharynx tissue equivalent.

Pro-Inflammatory Cytokine Production after Bacterial Exposure

For the bacterial particles, fluorescently labeled particles were addedto the tissue construct, at control, low, and high levels. Supernatantswere collected after a 24-h treatment and fluorescently labeled sampleswere visualized. Confocal microscopy was performed on heat-inactivatedand fluorescently labeled E. coli (Alexafluor 488)- and S. aureus(Alexafluor 594)-exposed mucosal constructs (FIG. 5).

Using Luminex™ technology, we simultaneously quantified the 22 cytokineand chemokines in the collected treatment media. This BioPlex kit usedis based on ELISA methods with beads coated with 22 antigens. Bacterialtreatment significantly increased IL-8 and IL-10, along with IL-1a,IL-12 (p40), GM-CSF and IFN-γ in almost all treatment conditions (FIG.6).

Example 13 Corticosteroid Treatment

Multiplex analysis of cytokine production along with treatment effectsof cellular proliferation were quantified. Surfactant production can beevaluated by, for example, ELISA and immunohistochemical stainingmethods.

Suppression of Cytokine Production by Corticosteroids

Having established that the constructs of the present invention could beimmunostimulated, we next demonstrated that they could beimmunosuppressed, by corticosteroids. Corticosteroids are the mostwidely prescribed drugs for allergic rhinitis and other respiratorydiseases that result in a mucosal inflammatory response. Generally,aerosolized local application of these anti-inflammatory medications isnon-problematic at levels that do not cause high systemic introduction.

The effects of dexamethasone and hydrocortisone were studied in the invitro alveolar artificial tissue constructs of the present invention todetermine whether the tissue responded to immunosuppressants. Theaddition of dexamethasone and hydrocortisone to the bilayer mucosalconstruct resulted in a significant decrease in cytokine production bythe native cells (FIG. 7).

The effect of immunosuppressant hormones was clearly observed withGM-CSF, eotaxin, MCP-1, IL-6, IL-8, and IL-12. The epithelial cellscultured in a flask format also showed a decrease in cytokine productioncompared to the control tissue with the addition of hormones (data notshown here). The particulate treatment of the epithelial cells in flaskformat induced a reduction in cytokine production. Different irritantconcentrations are being considered to relate inflammation to degree ofenvironmental particulate exposure.

Example 14 Tissue Functionality Evaluation: Cytochrome P450 Activity

The effects of polycyclic carcinogens on cytochrome P450 activity in theartificial tissue constructs was also analyzed. Cytochrome P450 proteinsare involved in the metabolism of many exogenous and endogenouschemicals, including drugs, steroids, and toxins. Cytochrome P450induction is indicative of chemical metabolism. Cellular P450 enzymeactivity of CYP1A1 & CYP2B 1 families was studied. The bilayerconstructs were prepared from biopsy samples of three differentpatients.

The mucosal construct was exposed to either 3-methylcholantherine andβ-nitrophenol (FIG. 8) for 24 h, followed by 7-ethoxy resorufin (5-15μM) or 7-pentoxy resorufin (5-15 μM) addition (for 30 min) to quantifyCYP 1A1 and CYP 2B1 activity, respectively. A β-glucuronidase andarylsulfatase enzyme cocktail was then added to the samples and theywere incubated for 2 h at 37° C. Fluorometric absorbance was thenmeasured. A standard curve was prepared using resorufin to quantify theP450 activity. 3-Methylcholanthere and β-nitrophenol are knownenvironmental carcinogens found in cigarette smoke and in airborneindustrial byproducts.

Our results showed that the alveolar artificial tissue bilayer constructexhibited p450 (CYP1A1 and CYP2B1) activity when exposed to polycycliccarcinogens. The alveolar artificial tissue construct cells wereactivated on exposure to 3-methylcholanthere, while the cells were lessactivated by BNF treatments (FIG. 8). These results show that thebilayer mucosal construct shows similar functionality to primary lungtissues.

Example 15 Surfactant Vesicles Cycling (Qualitative Measurements)

The visualization of surfactant vesicles in the epithelium bytransmission electron microscopy, described in example 3, qualitativelyshows that the presence and activity of Type II epithelial cells. Thiswas also demonstrated by FM-143FX uptake by the epithelium in themucosal bilayer construct; shown in example 7.

Example 16 Surfactant Components (Quantitative Measurements)

As examples, release of cytokines and release of lung surfactant by TypeII epithelial cells can be assessed, as manifestations of mucosalactivity. Cytokines in culture fluids can be measured by multiplexsystems. Lung surfactant components can be monitored using commerciallyavailable surfactant protein D (SP-D) ELISA kits. More sensitivebead-assisted BioPlex assays can also be used.

Detection of SP-D Using an ELISA Kit

An ELISA kit (AntibodyShop A/S, Denmark) was calibrated according to themanual, using internal standard solutions of SP-D. While the calibrationcurve remained linear at high concentrations of SP-D, significant dropof registered optical signal at concentrations below ˜3-4 ng/mLrestricted the practical sensitivity to ˜3 ng/mL.

Samples of culture fluid, ˜0.5 mL each, were obtained directly from thecultivation wells containing the artificial tissue construct samples,which had been grown for 2 weeks. Samples were serially diluted. TheELISA assay showed that the culture fluids contained less than 1 ng/mLSP-D. A higher sensitivity assay was needed.

We next fabricated SP-D-specific beads for the BioPlex. Thebead-assisted SP-D-detection was based on the principal of a sandwichimmunosorbent assay, where detection antibodies, coupled with biotin,were attached to a streptavidin-phycoerythrine (SA-PE) fluorescentcomplex.

A number of polyclonal and monoclonal anti-SP-D antibodies were assessedto assemble the bead-assisted SP-D detection tool. However, mostcombinations of available antibodies showed no visible detection of theSP-D taken from the standards of the SP-D ELISA kit. Of those tested,only a combination of MAB3132 as a capturing antibody, and detectionantibody taken from the SP-D demonstrated measurable sensitivity towardsSP-D. The reason for the poor sensitivity to SP-D may have been strongoverlapping of the epitopes with the available antibodies.

To demonstrate that the artificial tissue constructs of the presentinvention possess functional features similar to native mucosa,non-destructive detection of surfactant proteins was conducted. As atest of mucosal functionality, we examined modulation of surfactantrelease by cytokines and hormones, using non-destructive monitoring. Theclassic sandwich ELISA was not a useful tool for monitoring surfactantcomponents released by in vitro samples; its sensitivity wasinsufficient.

Abnova (Taiwan) sells recombinant SP-D fused with a GST tag, and anumber of anti-SP-D and anti-GST antibodies. One pair of theseantibodies showed sensitivity to SP-D-GST recombinant protein close to0.02 ng/mL in a sandwich ELISA. The explanation for this impressivesensitivity was apparently the use of a high-affinity anti-GST antibodyas the detection antibody. We examined a similar detection system inconditions of direct competition between SP-D-GST and natural SP-D. Thepresence of the latter in solution should block capturing the fusedSP-D-GST, thus eliminating the detection of the GST marker. While morecomplex and less convenient than a traditional ELISA, such a system candetect low concentrations of surfactant proteins released in the culturefluid.

Other methods for monitoring components of lung surfactant include PCRmeasurements of mRNA encoding various protein components of thesurfactant. This method is destructive, because it requires extractionof RNA from the cells. Immunohistochemical staining of sectioned samplesof the constructs can also be used to visualize several components inone run. It is sensitive and informative, but destructive, as ittypically requires fixation of the cells. Time-dependent measurements ofthe fluorescence produced by FM143-FX dye penetrating into lamellarbodies of Type II cells can also be used. The method can be employedsemi-non-destructively and is potentially very sensitive.

Example 17 Autologous Blood Compartment

In addition to a bilayer motif of the artificial tissue construct, anautologous blood compartment is added. The model comprises a bilayertissue engineered structure comprising a polarized epithelium, whichserves as a preliminary site of antigen interaction, followed by anendothelial layer, which serves as a barrier between the epithelium andthe neutrophil compartment. The in vitro model, the attachment andsubsequent migration of peripheral blood leukocytes through theendothelial layer, represents tissue vasculature, followed bytransepithelial migration resulting in cellular activation. Thecontribution of each leukocyte cell population can be dissected tobetter understand the contributing role of local tissue components.

Peripheral blood mononuclear cells (PBMCs) are isolated from whole bloodsamples. To minimize red blood cell contamination and removegranulocytes in the limited blood volume, the cell suspension isseparated using Histopaque-1077™ tubes. The Buffy coat interface iscollected and repeatedly washed in PBS. The resulting PBMC pellet isdisaggregated in cell culture medium containing ˜1% serum (˜80-95%lymphocytes, ˜5-20% monocytes; suspension concentration: ˜1×106 whiteblood cells per mL). Lymphocytes are isolated from the PBMC suspensionby plating the white blood cells on TC plates (˜1 h, 37° C., ˜5% CO₂),where monocytes adhere and lymphocytes stay in suspension.Centrifugation of the supernatant (˜200 g, ˜5 min) yields ˜99-100%lymphocyte population. Alternatively, monocytes are isolated from PBMCsusing MACS. The isolated PBMCs are incubated with anti-human CD14antibody-conjugated superparamagnetic microbeads. The labeledsuspensions is run through a MACS depletion column (yields ˜70-100%monocytes, ˜0-30% lymphocyte contamination; ˜88-100% purity isacceptable). B and T cells are isolated via negative selection usingMACs.

Example 18 Respiratory Syncytial Virus (RSV) and Influenza Infection ofMucosal Models

Respiratory Syncytial Virus (RSV)

The paramyxoviridae family of RNA viruses is characterized bynegative-sense single-stranded RNA genomes. Common respiratory humandiseases caused by paramyxoviridae viruses include respiratory syncytialvirus (RSV), and parainfluenza viruses. The F glycoprotein facilitatesfusion with cellular membranes and cell entry. Another relevant cellattachment protein that interacts with sialic acid moieties on the cellsurface is “G protein” (Harris & Werling (2003) Cell Microbiol. 5,671-80). Serum titers to RSV are found in the majority of children bythe age of two (Glezen et al. (1986) Am. J. Dis. Child. 140, 543-6;Chanock & Finberg (1957) Am. J. Hyg. 66, 291-300; Chanock et al. (1957)Am. J. Hyg. 66, 281-90). Primary RSV infection of the airway epitheliumlead to mucus overproduction and an inflammatory response of the tissueand lymphocytes (T cells, B cells, eosinophils, neutrophils) andresultant cytokine, chemokine, and leukotriene production (Becker (2006)Virus Genes 33, 235-52; Braciale (2005) Proc. Am. Thorac. Soc. 2, 141-6;Garofalo et al. (2001) J. Infect. Dis. 184, 393-9; Tripp et al. (2001)Cell Immunol. 207, 59-71; Tripp et al. (2000) Cytokine 12, 801-7; Trippet al. (2002) J. Infect. Dis. 185, 1388-94; McNamara et al. (2004b)Lancet 363, 1031-7; McNamara et al. (2005) J. Infect. Dis. 191, 1225-32;McNamara et al. (2004a) Eur. Respir. J. 23, 106-12; Falsey (2005) Exp.Lung Res. 31 (Suppl 1), 77; Piedimonte et al. (2005) Pediatr. Pulmonol.40, 285-91). Serious sequelae of RSV infection, including bronchiolitisand pneumonia, have been reported in children and the elderly (Collins &Graham (2008) J. Virol. 82, 2040-55; Shay et al. (1999) JAMA 282,1440-6; Falsey (2005) Exp. Lung Res. 31 (Suppl 1), 77; Ottolini &Hemming (1997) Drugs 54, 867-84; Wyde (1998) Antiviral Res. 39, 63-79).Reliable animal models of RSV infection and pathology remain elusive(Byrd & Prince (1997) Clin. Infect. Dis. 25, 1363-8).

Various therapeutic approaches have been explored for RSV infection. Aninitial vaccine effort entailed the use of formalin-inactivated virus,which actually exacerbated disease in a number of recipients (Hall(1994) Science 265, 1393-4; Dudas & Karron (1998) Clin. Microbiol. Rev.11, 430-9). Subunit RSV vaccines with modified G and F glycoproteinshave shown limited efficacy in children under the age of 12 and inpatients with chronic lung disease (Dudas & Karron (1998) Clin.Microbiol. Rev. 11, 430-9; Venkatesh & Weisman (2006) Expert Rev.Vaccines 5, 261-8). A live attenuated virus vaccine approach, employingreverse genetics, has shown some success after initial failures withattenuation and lack of temperature sensitivity (Collins & Murphy (2005)Proc. Am. Thorac. Soc. 2, 166-73). Protein-modified viral vaccinesexhibit stable phenotype but lacked attenuation; there was someimprovement with genetically engineered cpts/404/1030 (Dudas & Karron(1998) Clin. Microbiol. Rev. 11, 430-9; Whitehead et al. (1999a) J.Virol. 73, 871-7; Whitehead et al. (1999b) J. Virol. 73, 9773-80).Enhanced efficacy has been shown in passive immunization approaches withmonoclonal antibodies, such as Synagis (palivizumab) and its improvedderivative, motavizumab; however, little is known about these monoclonalantibodies that interact with F and G proteins and the pathways leadingto the induction of immunity (Weltzin (1998) Expert Opin. Investig.Drugs 7, 1271-83; Johnson et al. (1999) J. Infect. Dis. 180, 35-40;Johnson et al. (1997) J. Infect. Dis. 176, 1215-24).

Example 19 Palvizumab Evaluation

The artificial tissue constructs of the present invention can be used toevaluate, for example, palvizumab. Neutralizing IgGs are believed toplay a role in abrogating the transit of upper tract RSV infection tothe lower respiratory tract. These antibodies are directed towards the Fand G surface proteins. To study the efficacy of the anti-F protein mAbpalvizumab in our alveolar artificial tissue constructs, the mucosaltissue can be infected with the Long strain of RSV and then treated withpalvizumab, a commercially available monoclonal anti-RSV antibody.

The artificial tissue constructs can be infected with serial ofdilutions of the Long strain RSV for a minimum of ˜2 h at ˜37° C., usingfor example, up to a thousand-fold TCID₅₀ dilution of the virus. Afterremoval of excess virus, the artificial tissue construct can be culturedfrom ˜2 h up to ˜5 days. The replication of RSV can be determined by Fprotein expression, using, for example, ELISA-based methods. The tissueconstruct is then fixed and incubated with biotin-conjugated anti-Fprotein monoclonal antibody, and labeled with a secondary antibody,followed by a spectrophotometric read-out. The neutralizing titer isdefined as the antibody concentration that results in 50% or morereduction in the spectrophotometric read-out. RSV-infected cells will becompared against non-treated controls. The effects of RSV infection ofcellular viability can also be evaluated as this may affect themonoclonal antibody interaction. The cytokine profile of the infectedand non-infected cells before and after palvizumab treatment can also bequantified by, for example, multiplex analysis of the culturesupernatants. T cell functionality and proliferation can also beevaluated.

Example 20 Influenza

Despite considerable research efforts over recent decades, influenzavirus continues to have a significant impact on the human population,worldwide (Kitler et al. (2002) Vaccine 20 (Suppl 2) S5-14; Monto (2000)Am. J. Manag. Care 6 (5 Suppl) 5255-64). Common flu or influenza belongsto the Orthomyxoviridae family of enveloped, negative-sense segmentedRNA viruses, and influenza A, influenza B, and influenza C are thecommon strains. Although types A and B are responsible for most humaninfections, type A strains are considered more virulent and have beenthe primary focus of numerous therapeutic approaches for understandingand treating the disease. Type A influenza is defined by its twoimportant surface antigenic proteins; hemagglutinin, HA (16 subtypes)and neuraminidase, NA (9 subtypes). The virus uses HA and NA forattachment and movement through the respiratory mucosal membrane.Hemagglutinin binds to sialic acid, resulting in viral attachment andfusion. Neuraminidase is involved in removal of sialic acid moietiesthat allows the virus to move though the mucosal surfaces after pinchingoff from the cellular membranes. HA and NA are also the antigenic sitesof interaction with neutralizing antibodies that prevent viral infectionand replication. Due to its relatively high degree of mutability, theinfluenza viruses continue to undergo antigenic drift (e.g., mutation ofHA, NA surface proteins) and shift (due to its segmented RNA genomeallowing re-assortment of two viral genomes in the same host). Thesealterations in viral phenotype can result in the virus evading the hostimmune system and serving as the impetus for the annual reformulation ofthe trivalent influenza vaccine (Zambon (1999) J. Antimicrob. Chemother.44 (Suppl B), 3-9).

MedImmune's cold-adapted live-attenuated influenza vaccine FluMist® hasbeen shown to offer protective immunity against mis-matched vaccinestrains, suggesting a broader palette of immune response to this ‘live’vaccine. The efficacy of the vaccine has been suggested to be the resultof multiple attenuating effects on several genes (Jin et al. (2003)Virology 306, 18-24). This vaccine has been shown to be as effective asthe inactivated viral vaccine in healthy people (5 to 49 years of age).However, the mechanism(s) by which it induces protective mucosalimmunity remain(s) unclear.

Immunological Characterization with FluMist®

The in vitro respiratory mucosal models of the present invention can beused to evaluate, for example, FluMist®. The in vitro mucosalimmunological model of the present invention can be used to assess viraltransport processes as they relate to the efficacy of immune responseinduction. The mucosal tissue equivalent can be infected with thecold-adapted, temperature-sensitive live attenuated influenza trivalentFluMist® vaccine. To examine the immunological properties of theconstruct, the H1N1 strain in the FluMist® vaccine can be evaluated. Thereplication efficiency and delivery at the nasal passage temperature(˜25-28° C.), nasopharynx temperature (˜34° C.), and lung temperature(˜37° C.) can be studied to further evaluate the constructs. Resultsobtained can be compared with published clinical reviews on the efficacyof the vaccine in the clinical setting (Wiselka (1998) Vaccine Safety,Textbook of Influenza. Blackwell Sciences; Keitel et al. (2001) J.Infect. Dis. 183, 329-332).

The mucosal construct is infected with the H1N1 strain in FluMist® atthe median tissue culture infectious dose (TCID₅₀) administered topatients. The construct is placed in an incubator at 34° C. Thecorresponding control is placed at 37° C. FITC-labeled H1N1 viralantibodies (Chemicon Inc, Argene, Inc) are used to detect the presenceand location of the virus. For example, the degree of infection can bequantified by labeling the remaining viral particles in the inocula.Fusion events can be imaged by confocal microscopy. The intensity of thefluorescent signal is expected to increase once a fusion event andacidification occurs. The labeled antibody is introduced to the virallyinfected construct after a period of time. Alternatively, thefluorescent antibody is attached to the virus before exposure to thecells. Anti-MDV antibodies (Biodesign Inc.) can be used to detect thedegree of infection in a defined construct surface area. The infectedconstructs can be fixed with a gluteraldehyde-based fixative andembedded in an EPON-like polymer for high-resolution transmissionelectron microscopy imaging. Inflammation in the infected tissue can bequantified using, for example, ELISA-based methods. The multiplex beadarray Technique™ (BioPlex) can also be used to monitor cytokine levelsin small volume samples of culture fluids and donor sera. Culture mediafrom infected constructs can be used to quantify the cytokine response.The influenza virus can be inactivated with, for example,β-propiolactone (BPL) or similar chemicals to prepare the samples formetabolic testing.

A combination of functional assays can be used to determine the degreeof viral infection, neutralization and effective dose needed at varioustemperatures. For each donor, humoral immune responses to FluMist® inthe mucosal construct can be evaluated with, for example, ELISA- andBioPlex-based methods. Media collected at days 1, 4, 7, and 11 can beserially diluted and added to hemagglutinin-coated plates, followed byconjugation with a secondary antibody directed against theimmunoglobulin. Additionally, the Bioplex system, using beads coatedwith the specific hemagglutinin molecules in the FluMist® vaccine, candelineate the contribution of each vaccine component to the overallantibody response. Total IgA, IgG, and IgM can be quantified by, forexample, a sandwich ELISA technique using standard human serum for thestandard curve. Viral neutralization assays using collected media can beperformed by reintroduction to respiratory mucosal bilayer constructfollowed by, for example, FITC or another fluorescently labeledanti-influenza A monoclonal antibody. After treatment, the absence of afluorescent signal is indicative of viral neutralization and fluorescentcells indicate viral infection. Hemagglutination inhibition antibodytiters can be tested in the collected media by standard microtitermethods.

Primary epithelial and endothelial cells from various donors can betested to check the degree of infection with each of the FluMist®strains. A wider range of temperature, viral concentration and donor agemay be examined in the mucosal construct.

All documents, publication, manuals, article, patents, summaries,references and other materials cited herein are incorporated byreference in their entirety. Other embodiments of the invention will beapparent to those skilled in the art from consideration of thespecification and practice of the invention disclosed herein. It isintended that the specification and examples be considered as exemplaryonly, with the true scope and spirit of the invention being indicated bythe following claims.

What is claimed is:
 1. An artificial tissue construct comprising: (a) afirst cellular layer comprising alveolar primary epithelial cells andhaving a first face and a second face, (b) a second cellular layercomprising alveolar primary endothelial cells, and (c) either apopulation of B lymphocytes, or a population of B lymphocytes and Tlymphocytes, wherein the second layer is positioned in direct contactwith the first face or the second face of the first layer.
 2. Theartificial tissue construct of claim 1, wherein the alveolar primaryepithelial cells are human cells.
 3. The artificial tissue construct ofclaim 1, wherein the alveolar primary endothelial cells are human cells.4. The artificial tissue construct of claim 1, wherein the alveolarprimary epithelial cells and the alveolar primary endothelial cells arehuman cells.
 5. The artificial tissue construct of claim 4, wherein thehuman alveolar primary endothelial cells and the human alveolar primaryepithelial cells are from the same human.
 6. The artificial tissueconstruct of claim 1, further comprising primary alveolar macrophages.7. The artificial tissue construct of claim 6, wherein the primaryalveolar macrophages are interspersed among the alveolar primaryepithelial cells and the alveolar primary endothelial cells.
 8. Theartificial tissue construct of claim 6, wherein the primary alveolarmacrophages are interspersed among the alveolar primary epithelial cellsand the alveolar primary endothelial cells, and positioned between thefirst cellular layer and the second cellular layer.
 9. The artificialtissue construct of claim 1, further comprising a biocompatible membranepositioned between the first cellular layer and the second cellularlayer.
 10. The artificial tissue construct of claim 9, wherein thebiocompatible membrane is selected from the group consisting of basementmembrane, extracellular matrix, collagen, laminin, proteoglycan,vitronectin, fibronectin, poly-D-lysine and polysaccharide.
 11. Theartificial tissue construct of claim 9, wherein the biocompatiblemembrane is an extracellular matrix comprising laminin and collagen. 12.The artificial tissue construct of claim 1, further comprising bloodcells.
 13. The artificial tissue construct of claim 1, furthercomprising white blood cells.
 14. The artificial tissue construct ofclaim 4, further comprising blood cells.
 15. The artificial tissueconstruct of claim 4, further comprising white blood cells.
 16. Theartificial tissue construct of claim 6, further comprising blood cells.17. The artificial tissue construct of claim 6, further comprising whiteblood cells.
 18. The artificial tissue construct of claim 9, furthercomprising blood cells.
 19. The artificial tissue construct of claim 9,further comprising white blood cells.